Nosocomial (hospital-based) infections have become one of the most serious problems in infectious disease. Staphylococcus aureus is exceeded only by Escherichia coli as a leading cause of nosocomial infections. See, for example, Brumfitt, W. et al., Drugs Exptl. Clin. Res., 16:205-214 (1990). One type of S. aureus, methicillin-resistant S. aureus (MRSA), is of a particular interest because it is resistant to all penicillin-based antibiotics.
Patients in the intensive care unit are very susceptible to bacterial infections, due to interventions such as respiratory tubes and indwelling catheters. E. coli and S. aureus, if introduced into surgical wounds, the blood stream or the urinary tract, cause serious, sometimes life-threatening infections. The Infection Control Committees of most hospitals are constantly fighting this problem. There is no easy solution to the problem, but a partial solution in most xe2x80x9cnosocomial outbreaksxe2x80x9d is simply identifying the source of the infection. That is, is the infectious agent coming from a common source (e.g. an infected nurse or doctor, or an instrument such as a respirator) or is there some other reason for the sudden emergence of a single type of bacterial infection.
Hospital laboratories can quickly identify the infectious agent (e.g., S. aureus), but they do not have the ability to determine whether a single strain of the organism is causing the outbreak (and therefore a possible common source) or if several different strains are responsible for the outbreak. At the present time most of these outbreaks can only be characterized in retrospect, since the outbreak is over before the bacterial isolates can be identified. For example, in an outbreak of S. aureus in a hospital nursery, the isolates can be identified in a matter of 1 or 2 days, but the strain identification usually takes weeks or even months, because the strains are still analyzed by slow culture-based methods which are labor intensive.
Current methods of strain typing bacteria include phage typing, plasmid analysis, and antibiotic susceptibility (biotyping). See, for example, Zuccarelli, A. et al.: J. Clin. Microbiol., 28:97-102 (1990); Tokue, Y. et al.: Tohokin J. Exp. Med., 163:31-37 (1991); Coia, J. et al.: J. Med. Microbiol., 31:125-132 (1990); Pennington, T. et al.: J. Clin. Microbiol., 29:390-392 (1991); Tveten, Y. et al.: J. Clin. Microbiol., 29:110-1105 (1991); Fluit, A. et al.: Eur. J. Clin. Microbiol. Infect. Dis., 9:605-608 (1990); Thomson-Carter, F. et al.: J. Gen. Microbiol., 135:2093-2097 (1989); and Preheim, L. et al.: Eur. J. Clin. Microbiol. Infect. Dis., 10:428-436 (1991). These methods, currently used by the Centers for Disease Control, are laborious, time consuming (approximately one month) and often yield inconclusive results.
Consequently, there is a serious need in the medical community for means to not only identify the infectious agents, but also to rapidly characterize the strain or strains involved so that effective measures may be timely employed.
In brief, the present invention alleviates and overcomes certain of the above-mentioned drawbacks of the present state of the art through the discovery of novel methods and kits for rapidly fingerprinting DNA to identify prokaryotic and eukaryotic species, subspecies, and especially strains or individuals of the subspecies. As to prokaryotic organisms, the present invention is especially suited for identifying different bacterial strains involved in, for example, nosocomial infections, since the methods and kits are believed to be sensitive enough to detect differences between, for example, bacterial isolates of the same species. With respect to eukaryotes, the present invention contemplates identifying, for instance, species, subspecies, and the differences between the individuals of the subspecies, such as pedigrees.
Generally speaking, the present invention involves the use of polymerase chain reaction (PCR) technology and restriction fragment length polymorphism analysis of genomic DNA preferably containing numerous gene clusters, such as ribosomal RNA (rRNA) gene clusters. In accordance with the present invention, a specific DNA fragment in a gene cluster region, such as the rRNA intergene region, is amplified by PCR using two universal oligonucleotide primers. This area of the genome is generally ideal for such a procedure because a highly variable spacer region is flanked by two highly conserved genes which may be used as primer sites. Before PCR amplification, however, the oligonucleotides are preferably fluorescently labeled. While it is preferable to label the 5xe2x80x2 end primers, the 3xe2x80x2 end primers or both the 5xe2x80x2 and 3xe2x80x2 end primers, as well as the individual nucleotides utilized during PCR may be labeled. Because the 5xe2x80x2 end of each cluster is preferably flourescently-labelled, each fragment is represented as an individual peak on a waveform pattern. Therefore, since E. coli has seven clusters within its genome, multiple peaks will be present on the waveform pattern. The labeled PCR product may then be cleaved with a variety of restriction endonucleases and electrophoresed on an automated DNA sequencer.
Thus, the methods and kits of the present invention generally depend upon rapid, semiautomated DNA analysis, and more particularly, upon a type of DNA fingerprinting of multiple segments of DNA, such as the ribosomal RNA gene clusters, that are common to particular prokaryotic or eukaryotic species. Morover, the methods and kits are believed to be most beneficial in a clinical laboratory because they allow for rapid strain identification of pathogenic bacteria. The DNA fingerprinting methods and kits of the present invention are also believed to be more definitive than currently practiced methodologies, since genomic DNA is used.
One main advantage of the methods and kits of the present invention is the speed with which results are obtained. A preliminary screen by agarose gel electrophoresis of a PCR product can be completed within five to six hours after receiving a sample, such as a hospital isolate. More particularly, the differences in the intergene region are detected on, for example, a 2% agarose gel, by banding patterns following PCR. Identical strains will exhibit similar banding patterns, whereas strains from other sources or of different types will differ in band intensity and fragment size (as demonstrated in FIG. 1).
The preliminary screen can then be confirmed in approximately 24 hours by restriction fragment length polymorphism (RFLP) analysis on an automated sequencer. For example, more definitive analyses are performed on the Applied Biosystems, Inc. (Foster City, Calif.) vertical electrophoresis unit with the Genescan software. Following PCR, restriction endonuclease digestions are performed and the cleaved fragments are loaded directly onto the electrophoresis unit. Although a multitude of restriction endonucleases are available for performing this assay, HhaI and HindIII are believed to be the most informative concerning at least prokaryote typing.
MRSA digested with HhaI consistently exhibit patterns with fragment lengths of 276, 278, 283, and 299 base pairs (see FIG. 2). Although fragment sizes are consistent among strains, a remarkable difference in band intensity is noted for each individual strain. Band intensity is measured by the software as peak area and may be converted to gene dosage (gene copy number) by using a reference gene during the electrophoresis run. It is our belief that differences between strains or individuals of subspecies may be determined based on gene dosage of each fragment at these given positions. A gene which may be universal among eubacteria and may be useful as a standard gene for calculating gene dosages has also been identified. This gene is dna A.
HindIII restriction endonuclease digestions of MRSA have been used to corroborate HhaI data. Typical HindIII patterns exhibit two to three fragment groupings with varying fragment lengths (see FIG. 3). Each grouping is typically positioned fourteen to twenty bases away from its neighboring group. Strain determinations are based on the number of groupings present and fragment length. The speed of these procedures provides, for example, infection control personnel adequate information to contain and prevent the spread of nosocomial infections, rather than having analyses done retrospectively.
Accordingly, it can now be appreciated that the present invention is believed to provide a solution to identifying differences between species, subspecies, and strains of prokaryotic organisms and individuals of subspecies of higher life forms.
The above features and advantages will be better understood with reference to the FIGS., Examples, and Detailed Description set out herein below. It will also be understood that the methods and kits of the present invention are exemplary only and are not to be regarded as limitations of this invention.